Arachidonic acid has been shown to stimulate lung surfactant secretion from alveolar epithelial type II cells. To identify the (phospho)lipases responsible for generating arachidonic acid during lung surfactant secretion, the effects of various (phospho)lipase inhibitors on phosphatidylcholine (PC) secretion from rat alveolar type II cells were investigated. N-(p-amylcinnamoyl)anthranilic acid (ACA), a general inhibitor of phsopholipase A2 (PLA2), inhibited ATP-stimulated PC secretion in a dose-dependent manner. ACA also blocked PC secretion from type II cells stimulated by other secretagogues including phorbol 12-myristate 13-acetate, Ca2+ ionophore A23187 and terbutaline, indicating that PLA2 acts at a late step distal to the generation of second messengers. To determine which PLA2 isoform(s) is involved in lung surfactant secretion, selective inhibitors to different types of PLA2 were used to inhibit PLA2 activity in type II cells. The cytosolic PLA2 (cPLA2) inhibitor, arachidonyl trifluoromethyl ketone, was found to inhibit ATP-stimulated PC secretion, whereas the secretory PLA2 inhibitors, oleoyloxyethylphosphocholine, aristolochic acid, or p-bromophenacyl bromide, and the Ca2+-independent PLA2 inhibitors, palmitoyl trifluoromethyl ketone, or haloenol lactone suicide substrate, had no effect. In addition to PLA2, arachidonic acid is released from diacylglycerol (DAG) by DAG and monoacylglycerol lipases. The DAG lipase inhibitor, RHC-80267 also blocked ATP-stimulated PC secretion. The results suggest that both pathways for generating arachidonic acid via cPLA2 and DAG lipase may participate in lung surfactant secretion.